| Hydrogels based on genetically engineered polypeptide have a wide application prospect as a new biomaterial in biomedical fields due to their superior biocompatibility and tunable structure. This paper is mainly consisted of the fabrication of polypeptide-based hydrogels and their applications as the scafford in three-dimentional (3 D) cell culture and tissue engineering research; the preparation of polypeptide-based microgels to build porous cell-laden hydrogels by self-assembly and their applications for tissue engineering research; the preparation of PC10A(RGD)-QDs (P and A are coiled-coil sequences, C10 is a random coil sequence) hybrid nanogels and using for the targeted bioimaging research; using differently charged polypeptides as surface-ligand to modify QDs and their applications for the targeted bioimaging research. Specific works are as follows:(1) Photo-cross-linkable physical hydrogels based on polypeptide P and poly(ethylene glycol) diacrylate (PEGDA) were designed and synthesized, and their applications as the scaffold for 3 D cell culture and tissue engineering research.The polypeptide (Pcys or RGDPcys) was modified with PEGDA to form photosensitive macromer P-PEG-acrylate/RGDP-PEG-acrylate via the Michael-type addition reaction between the cysteine residues and acrylates. Photosensitive macromer can form hydrogels under the condition of photoinitiator 1-2959 and UV light. Both of the denatured experiment and rheological test confirmed that these gels are physical hydrogels. Rheological test also indicated that these hydrogels have high mechanical strength and tunable elasticity modulus. These hydrogels have good swelling property and stability, for example the swelling ratio and stability of 10% w/v RGDP-PEG-acrylate6k hydrogels are 38% and 15 days, respectively. In addition, these physical hydrogels have a good self-healing ability which can direct the self-assembly of microgels. The cytotoxicity test showed that these hydrogels have an excellent biocompatibility. Two-dimensional (2 D) cell growth experiment suggested that the surface cellular adhesion of polypeptide-based hydrogels could be regulated by changing the polypeptide composition (such as the introduction of cell integrin RGD).3 D cell culture test revealed that cells could remain high viability within these physical hydrogels, and could spread and migrate after 26 h. These results will provide a theoretical guidance for the applications of polypeptide-based hydrogels in tissue engineering.(2) Porous cell-laden hydrogels were builded by the bottom-up assembly of cell-laden microgels based on the self-healing property, and used for tissue engineering research.Microgels and cell-laden microgels were prepared by photolithography on the basis of part (1). The dynamic self-assembly of polypeptide on the surface of microgels directed the assembly of microgels to build porous cell-laden hydrogels. The size and shape of microgels could be regulated through photomask, and the height of microgels could be adjusted by controlling the height of spacer. In addition, these microgels could be assembled by the way of bottom-up and layer-by-layer. Compared assembled hydrogels with unassembled hydrogels, we found that these assembled hydrogels have larger pore size and better pore connection which are suitable for the input of nutrients and oxygen and the output of metabolites to insure the high cell viability in depth of assembled hydrogels.3 D cell culture experiment showed that cells within hydrogels could remain high activity after the fabrication and assembly of microgels, and could freely spread after 24 h. Therefore this material, synthetic method and assembly way of microgels are very suitable for imitating and building "tissue structure".(3) PC10A nanogels were used as vehicles to encapsulate and modify the hydrophobic fluorescent QDs, and applied for the targeted bioimaging research.PC10A nanogels were prepared by the method of "thermal denaturation-renaturation" dealing with low concentration of polypeptide PC10A aqueous solution. The TEM result showed that the size of nanogels could be regulated from 20nm to 300nm by changing the polypeptide concentration. The TEM and EDXS results indicated that hydrophobic QDs have been uniformly dispersed within nanogels, and the capacity of QDs within nanogels has tunability and saturability. Dynamic light scattering analysis showed that nanogels have a good stability (above 20 days), and the stability increases after loading with hydrophobic QDs. Fluorescent spectrum analysis showed that the hydrophobic QDs still have a good fluorescent intensity after being encapsulated into nanogels (quantum yield QY=33.5%). In addition, the fluorescent intensity increased with the increase of pH value. MTT result indicated that single PC10ARGD nanogels have no cytotoxicity, PC10ARGD-QDs nanogels with moderate amount of QDs still have a good biocompatibility, while PC10ARGD-QDs nanogel with excess amount of QDs have a high cytotoxicity. Laser scanning confocal imaging showed that PC10ARGD nanogels could endow hydrophobic QDs with a good targeting. Therefore, these nanogels are suitable as vehicles for encapsulating and modifying hydrophobic nanoparticles and endowing them with excellent biocompatibility, water stability and targeting, and have a great potential application in the cellular targeted imaging field.(4) Differently charged polypeptides were used to modify hydrophobic QDs and applied in the targeted bioimaging of tumor cells.Negatively charged polypeptide A, positively charged polypeptide B, polypeptides ARGD and BRGD were used as surface-ligands to modify CdSe@ZnS for preparing QD-A, QD-B, QD-BA (nearly neutral charged), QD-ARGD, and QD-BRGD probes. Agarose gel electrophoresis, size and zeta potential experiments had verified the successful preparation of differently charged fluorescent peobes. The size and charge of QD-polypeptide can be tuned by assembling with different polypeptides. Capillary electrophoresis (CE) test showed that the saturated molar ratio of polypeptide and QDs is 30:1. Fluorescent spectrum analysis suggested that QD-polypeptide probes have about 2 to 3-fold luminescence increase after being modified, and the luminescence increase has no obvious relationship with the charge of polypeptide. MTT result indicated that single polypeptide or QD-polypeptide has no acute cytotoxicity, while QD-ARGD has a certain chronic cytotoxicity. Therefore, this kind of probe is suitable for short-term cellular fluorescent imaging. Laser scanning confocal imaging showed that positively charged polypeptide B can promote the nonspecific cellular uptake of probe, while negatively charged A can suppress the nonspecific cellular uptake of probe. In addition, negative charge and targeted molecule have a synergistic effect on the targeted cellular uptake of probe. Polypeptide ARGD containing both negative charge and targeted molecular can effectively improve the targeted cellular uptake of probe. The result of flow cytometry experiment was consistent with the result of laser scanning confocal imaging. These results demonstrated that the QD-ARGD probe with high specific cellular uptake, high fluorescent intensity, and low background noise is expected to have a great potential application in the cellular targeted bioimaging. |
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